A method for breeding disease-resistant pepper by tissue culture

Abstract

The present application relates to the technical field of pepper tissue culture breeding, and discloses a pepper tissue culture breeding method with strong disease resistance, which comprises the following steps: taking cotyledon of pepper plant material as explant, performing surface disinfection treatment, inoculating into primary culture medium for culture, obtaining aseptic explant, transferring the aseptic explant into disease resistance induction type solid culture medium for induction culture, obtaining regenerated buds, sequentially performing recovery culture, rooting culture and seedling raising treatment, and transplanting into a cultivation substrate for continuous culture to obtain pepper regenerated plants; the disease resistance induction type solid culture medium comprises 1xMS basic culture medium, sucrose, PEG-4000, salicylic acid, potassium silicate, ammonium sulfate and agar powder, and the pH is 5.6-5.8. Through the cooperation of PEG-4000, salicylic acid, potassium silicate and ammonium sulfate, the browning and oxidation damage in the induction culture process of the explant are reduced, the formation of regenerated buds and rooting are promoted, and the acquisition of pepper regenerated plants and the disease resistance effect are improved.

Description

Technical Field

[0001] This invention relates to the field of chili tissue culture technology, specifically to a method for tissue culture of chili peppers with strong disease resistance. Background Technology

[0002] Chili peppers are an important vegetable crop. Tissue culture breeding can utilize explants to complete callus formation, bud differentiation, rooting, and regeneration of plants under sterile conditions, and can be used for chili pepper germplasm propagation and breeding material acquisition. Current chili pepper tissue culture is usually based on MS basal medium, with the addition of sucrose, solidifying agents, and plant growth regulators. However, browning, oxidative damage, and unstable regeneration status are still prone to occur during explant induction culture, which affects the formation of regenerated buds, rooting, and the acquisition of normal regenerated plants. Furthermore, the disease resistance of the obtained regenerated plants still has room for further improvement.

[0003] In the current culture medium preparation process, the components are usually mixed, pH adjusted, sterilized and cooled according to a predetermined formula. However, there is a lack of further control over the order of addition of components such as PEG-4000, salicylic acid, potassium silicate and ammonium sulfate, as well as the temperature treatment after high-pressure steam sterilization. This can easily cause rapid changes in the salicylic acid content and the soluble silicon content (calculated as SiO2) during the culture process, which in turn affects the degree of browning of explants, H2O2 content, MDA content, PPO activity and subsequent regeneration status. At the same time, directly cooling to form a solid culture medium after sterilization is not conducive to maintaining a suitable state of salicylic acid and soluble silicon content changes in the culture medium during subsequent culture.

[0004] Furthermore, the tissue culture process for chili peppers typically involves sequential stages of primary culture, induction culture, recovery culture, rooting culture, hardening-off treatment, and transplanting. The culture temperature, light duration, darkness duration, light intensity, and culture time at each stage directly affect explant survival, callus formation, bud differentiation, rooting, and the acquisition of regenerated chili pepper plants. However, existing methods still have shortcomings in the coordination between culture medium composition, culture medium preparation methods, and each culture stage, making it difficult to simultaneously achieve low browning of explants, good regeneration performance, and disease resistance of the resulting regenerated chili pepper plants. Therefore, how to reduce browning and oxidative damage during the culture of chili pepper explants, improve the formation of regenerated buds, rooting, and the acquisition of regenerated chili pepper plants, and enhance the disease resistance of regenerated chili pepper plants through the synergistic control of culture medium composition, culture medium preparation process, and tissue culture conditions is a problem that needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for tissue culture breeding of chili peppers with strong disease resistance. This method solves the problems of browning and oxidative damage of explants, limited regeneration of shoots and rooting, low rate of obtaining normal regenerated plants, insufficient coordination of culture medium components and preparation process, and the need to improve the disease resistance of the obtained regenerated chili pepper plants.

[0006] To address the above problems, the present invention provides the following technical solution: This invention provides a method for tissue culture of chili peppers with strong disease resistance, using the following technical solution: A method for tissue culture of disease-resistant chili peppers, comprising the following steps: Cotyledons of chili plants were selected as explants. The explants were surface disinfected and inoculated into a primary culture medium under aseptic conditions to obtain sterile explants. The sterile explants were transferred to a disease-inducing solid culture medium for induction culture to obtain regenerated shoots; The regenerated buds were transferred to a recovery culture medium for recovery culture to obtain regenerated buds after recovery culture. The regenerated shoots after recovery culture were transferred to a rooting medium for rooting culture to obtain pepper tissue culture seedlings; The chili tissue culture seedlings were hardened off and then transplanted into a cultivation substrate for further cultivation to obtain regenerated chili plants. The disease resistance inducing solid culture medium comprises 1×MS basal medium, sucrose at a mass-volume concentration of 2.5%–3.5%, PEG-4000 at a mass-volume concentration of 2.0%–4.0%, salicylic acid at a mass-volume concentration of 0.0015%–0.0025%, potassium silicate at a mass-volume concentration of 0.010%–0.015% (based on SiO2), ammonium sulfate at a mass-volume concentration of 0.066%–0.396%, and agar powder at a mass-volume concentration of 0.6%–0.8%. The pH of the disease resistance inducing solid culture medium is 5.6–5.8.

[0007] By employing the above technical solution, PEG-4000, salicylic acid, potassium silicate, and ammonium sulfate are added to the basic culture conditions consisting of 1×MS basal medium and sucrose, so that the sterile explants are in a culture environment containing PEG-4000, salicylic acid, and a silicon source during the induction culture stage. When PEG-4000 and salicylic acid are present together, the increase in H2O2 content, MDA content, PPO activity, and explant browning rate during culture under conditions where salicylic acid is added alone can be reduced; potassium silicate can provide soluble silicon for the disease resistance induction solid medium.

[0008] PEG-4000, salicylic acid, potassium silicate, and ammonium sulfate are present in the aforementioned concentrations in the disease-inducing solid culture medium. This ensures that the salicylic acid content and the soluble silicon content (calculated as SiO2) remain relatively constant during cultivation, reducing browning and oxidative damage in pepper explants and improving bud differentiation, rooting, and the acquisition of normal regenerated plants. The regenerated pepper plants, after inoculation with pepper anthracnose fungus, exhibit lower disease index and incidence, thus yielding pepper regenerated plants with good anthracnose resistance.

[0009] Preferably, the disease resistance-inducing solid culture medium is prepared according to the following method: Add deionized water according to the final culture medium volume, then add 1×MS basal medium and sucrose and stir to obtain the basal culture medium; The basic culture medium was heated and kept at a constant temperature. PEG-4000 and salicylic acid were added and stirred to obtain the culture medium. Maintain the temperature of the culture medium, and add an aqueous solution of potassium silicate as the form of potassium silicate addition to the culture medium, wherein the amount of potassium silicate is calculated according to the mass of SiO2 contained in the aqueous solution of potassium silicate; After the potassium silicate aqueous solution is added, the pH of the culture medium is adjusted using hydrochloric acid aqueous solution. Maintain the temperature of the culture medium, add ammonium sulfate to the culture medium and stir; Add deionized water to the culture medium after adding ammonium sulfate to make up the volume, then add agar powder and stir. The culture medium after adding the agar powder is dispensed into tissue culture containers, and the culture medium in the tissue culture containers is sterilized by autoclaving. After high-pressure steam sterilization, the culture medium in the tissue culture container is cooled and isothermally incubated. After isothermally incubation, the culture medium in the tissue culture container is allowed to cool naturally at room temperature to obtain the disease-inducing solid culture medium.

[0010] By adopting the above technical solution, 1×MS basal medium and sucrose are first added to deionized water to form the basal culture medium. Then, PEG-4000 and salicylic acid are added to the basal culture medium for dispersion. Subsequently, potassium silicate aqueous solution is added and pH is adjusted. Then, ammonium sulfate is added so that PEG-4000, salicylic acid, potassium silicate and ammonium sulfate are added to the culture medium in a determined order.

[0011] Compared to the method of simultaneously adding PEG-4000, salicylic acid, potassium silicate, and ammonium sulfate to the basal culture medium and then uniformly adjusting the pH, the stepwise addition method described above allows the salicylic acid content and soluble silicon content (calculated as SiO2) to maintain different changes during the culture process, corresponding to a lower explant browning rate and higher regeneration, rooting, and normal regeneration plantlet acquisition indicators. After autoclaving, isothermal incubation allows the culture medium to undergo a defined temperature maintenance process before naturally cooling to form a solid culture medium. This helps maintain the salicylic acid content and soluble silicon content (calculated as SiO2) during the culture process and improves the subsequent regeneration culture status of pepper explants.

[0012] Preferably, when preparing the basic culture medium: Based on the final culture medium volume, the deionized water accounts for 75% to 80% of the final culture medium volume; The stirring speed after adding the 1×MS basal medium and the sucrose was 100-150 rpm.

[0013] By adopting the above technical solution, 1×MS basal medium and sucrose are added to deionized water accounting for 75% to 80% of the final culture medium volume, and stirred at 100 to 150 rpm. This ensures that the 1×MS basal medium and sucrose are dispersed before the subsequent addition of PEG-4000, salicylic acid, potassium silicate aqueous solution and ammonium sulfate. At the same time, this leaves room for volume adjustment for the subsequent addition of deionized water to make up the volume, so that the mass and volume concentration of each component in the culture medium can be controlled according to the final culture medium volume.

[0014] Preferably, when adding the PEG-4000 and the salicylic acid to the basic culture medium: The basic culture medium is heated to 45–55°C and kept at a constant temperature. After adding the PEG-4000 and the salicylic acid, stir continuously at 150-200 rpm for 25-35 min.

[0015] By employing the above-mentioned technical solution, PEG-4000 and salicylic acid are added to the basic culture medium at 45–55°C, and the mixture is continuously stirred at 150–200 rpm for 25–35 minutes to ensure that PEG-4000 and salicylic acid are dispersed before the addition of potassium silicate aqueous solution. When PEG-4000 and salicylic acid coexist in the culture medium, they can reduce the H2O2 content, MDA content, PPO activity, and browning degree of pepper explants during culture, thereby mitigating oxidative damage to pepper explants during culture.

[0016] Preferably, when adding the potassium silicate aqueous solution to the culture medium and adjusting the pH: When the potassium silicate aqueous solution is added, the temperature of the culture medium is maintained at 45-55°C; The potassium silicate aqueous solution is added over a period of 5 to 10 minutes. After the potassium silicate aqueous solution is added, the pH of the culture medium is adjusted with 1 mol / L hydrochloric acid aqueous solution for 10-15 min.

[0017] By employing the above technical solution, the potassium silicate aqueous solution is added to the culture medium within 5-10 minutes at 45-55℃. After the addition of the potassium silicate aqueous solution, the pH of the culture medium is adjusted using a 1 mol / L hydrochloric acid aqueous solution, ensuring that the addition of potassium silicate and the pH adjustment of the culture medium are performed in a predetermined order. Potassium silicate provides a source of soluble silicon in the culture medium, ensuring that the resulting disease-resistant inducing solid culture medium maintains a corresponding soluble silicon content (calculated as SiO2) during the culture process, which corresponds to the regeneration of pepper explants and the acquisition of normally regenerated plants.

[0018] Preferably, when adding the ammonium sulfate to the culture medium: The temperature of the culture medium is maintained at 45–55°C; After adding the ammonium sulfate, adjust the stirring speed to 100-150 rpm and continue stirring for 12-18 minutes.

[0019] By adopting the above technical solution, after completing the addition of PEG-4000, salicylic acid and potassium silicate and pH adjustment, ammonium sulfate is added to the culture medium at 45-55℃, and stirring is continued at 100-150 rpm for 12-18 min to fully disperse the ammonium sulfate in the culture medium. The ammonium sulfate is added to the culture medium after the potassium silicate is added and the pH is adjusted, thereby maintaining the predetermined order of addition of the culture medium components.

[0020] Preferably, during the addition of the agar powder, the high-pressure steam sterilization, and the isothermal insulation: Add deionized water to the culture medium after adding ammonium sulfate to make up the volume, then add the agar powder, and stir at 100-150 rpm after adding the agar powder; When the culture medium dispensed into the tissue culture container is subjected to high-pressure steam sterilization, the high-pressure steam sterilization temperature is 121°C, the pressure is 0.1 MPa, and the time is 20-25 min. After the high-pressure steam sterilization is completed, the culture medium in the tissue culture container is cooled to 68-72°C and isothermally incubated at 68-72°C for 45-60 minutes.

[0021] By adopting the above technical solution, after adding ammonium sulfate, deionized water is used to make up the volume, then agar powder is added and stirred to ensure that the components in the culture medium reach the concentration determined according to the final culture medium volume, forming a culture solution that can gel upon cooling. The culture solution is then autoclaved at 121°C and 0.1 MPa for 20–25 minutes. After sterilization, the culture solution is cooled to 68–72°C and isothermally held for 45–60 minutes, allowing the culture solution to undergo a predetermined temperature holding process before naturally cooling to form a solid culture medium.

[0022] Compared with direct cooling to room temperature after high-pressure steam sterilization, the above-mentioned isothermal insulation can maintain different changes in salicylic acid content and soluble silicon content (calculated as SiO2) during the cultivation process, which corresponds to a higher rate of normal regenerated plants.

[0023] Preferably, when the cotyledons of the pepper plant material are selected as the explants and the explants are inoculated into the primary culture medium: When the cotyledons of the pepper plant material are selected as the explant, the length of the explant is 8-12 mm; When the explants are inoculated into the primary culture medium under aseptic conditions, the number of explants inoculated is 30.

[0024] By employing the above technical solution, the length of the explants is controlled to be 8–12 mm, and 30 explants are inoculated at a time, ensuring that the pepper explants entering the primary culture and subsequent induction culture have the appropriate tissue size and inoculation quantity. After surface sterilization and primary culture, sterile explants are obtained, providing sterile plant tissue material for subsequent transfer to the disease-resistant inducing solid culture medium for induction culture.

[0025] Preferably, when transferring the sterile explants to the disease-inducing solid culture medium for induction culture: The sterile explants were transferred to the disease resistance-inducing solid culture medium for induction culture at a temperature of 23–27°C, with a daily light duration of 14–16 h and a dark duration of 8–10 h, and the daily light and dark duration totaled 24 h. The light intensity was 2000–3000 lx, and the culture time was 14–28 days.

[0026] By adopting the above technical solution, the sterile explants are cultured in the disease resistance inducing solid medium for 14-28 days under the conditions of 23-27℃, 14-16h light per day, 8-10h darkness per day, and light intensity of 2000-3000lx. This allows the sterile explants to complete callus formation and bud differentiation and obtain regenerated buds in the culture environment provided by the disease resistance inducing solid medium, while maintaining the survival status of the explants and providing regenerative material for subsequent recovery and rooting culture.

[0027] Preferably, when performing the recovery culture on the regenerated buds, the rooting culture on the regenerated buds after recovery culture, and the hardening-off treatment on the pepper tissue culture seedlings: When the regenerated buds are transferred to the recovery culture medium for recovery culture, the recovery culture temperature is 23-27°C, the daily light time is 14-16 hours, the daily dark time is 8-10 hours, and the daily light and dark time is 24 hours, and the culture time is 10-20 days. When the regenerated shoots after the recovery culture are transferred to the rooting medium for rooting culture, the rooting culture temperature is 23-27℃, the daily light time is 14-16h, the dark time is 8-10h, and the culture time is 15-25d. When the chili tissue culture seedlings are subjected to the hardening treatment, the hardening temperature is 23-27℃ and the hardening time is 5-10 days.

[0028] By adopting the above technical solution, the regenerated buds are sequentially subjected to recovery culture and rooting culture, so that the regenerated buds that have undergone induction culture continue to grow and form a complete root system, thereby obtaining the chili tissue culture seedlings; then the chili tissue culture seedlings are subjected to hardening treatment, and the chili tissue culture seedlings that have completed the hardening treatment are transplanted into the cultivation substrate for continued cultivation, so that the chili tissue culture seedlings complete the transition from tissue culture conditions to cultivation conditions, thereby obtaining the regenerated chili plants.

[0029] The process involves induction culture, recovery culture, rooting culture, hardening treatment, and transplanting, which allows the pepper explants to complete a continuous culture process from sterile explants to regenerated pepper plants. The resulting regenerated pepper plants exhibit good resistance to pepper anthracnose.

[0030] This invention provides a method for tissue culture of disease-resistant chili peppers. It has the following beneficial effects: 1. This invention reduces browning of explants during induction culture by adding 1×MS basal medium, sucrose, PEG-4000, salicylic acid, potassium silicate, ammonium sulfate and agar powder to a disease resistance induction solid medium and controlling the pH, thereby promoting the formation of regenerated shoots and rooting culture, and improving the acquisition of pepper tissue culture seedlings, the acquisition of regenerated pepper plants and the disease resistance effect.

[0031] 2. This invention involves sequentially adding 1×MS basal medium, sucrose, PEG-4000, salicylic acid, potassium silicate aqueous solution, ammonium sulfate, and agar powder, followed by isothermal incubation after autoclaving. This allows PEG-4000, salicylic acid, potassium silicate, and ammonium sulfate to enter the culture medium in sequence, reducing explant browning and improving the formation of regenerated shoots, rooting culture, and the acquisition of regenerated pepper plants.

[0032] 3. This invention selects cotyledons of chili plant material as explants and sequentially carries out primary culture, induction culture, recovery culture, rooting culture and hardening treatment. The temperature, light time, dark time and culture time of each culture stage are controlled so that the explants can complete the process of regenerating buds, obtaining chili tissue culture seedlings and transplanting them into the cultivation substrate for continued culture, thereby improving the acquisition of regenerated chili plants. Attached Figure Description

[0033] Figure 1 This is a curve showing the change in the regeneration state of chili pepper tissue culture according to the present invention; Figure 2 The UV-Vis absorption curve of the receiving solution of the culture medium of this invention; Figure 3 The UV-Vis absorption curves of the MDA colorimetric reaction solution of explants from different treatment groups cultured for 7 days are shown below. Figure 4 The figures show the curves of changes in salicylic acid and soluble silicon content during the cultivation of different treatment groups according to the present invention; wherein, A is the curve of change in salicylic acid content and B is the curve of change in soluble silicon content. Figure 5 The visible near-infrared reflectance spectrum curves of leaves of regenerated pepper plants in different treatment groups after inoculation with anthracnose fungus are shown. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0036] MS basal medium is plant tissue culture grade Murashige and Skoog (MS) basal medium, containing macro-elements, micro-elements, iron salts and vitamin components of the standard MS formulation, and is prepared according to the standard 1×MS concentration when used.

[0037] Sucrose, CAS No. 57-50-1, molecular formula C 12 H 22 O 11 Analytical purity.

[0038] Agar powder, CAS No. 9002-18-0, plant tissue culture grade, gel strength not less than 800 g / cm³ 2 .

[0039] Polyethylene glycol 4000 (PEG-4000), CAS No. 25322-68-3.

[0040] Salicylic acid, also known as 2-hydroxybenzoic acid, CAS number 69-72-7, molecular formula C7H6O3.

[0041] Potassium silicate, CAS No. 1312-76-1, industrial grade, is a water-soluble potassium silicate system. The modulus is expressed as the molar ratio of SiO2 to K2O, with a modulus of 2.5-3.0. The amount of potassium silicate in the culture medium is calculated based on the mass of SiO2 contained in the aqueous solution.

[0042] Ammonium sulfate, CAS No. 7783-20-2, molecular formula (NH4)2SO4, analytical grade.

[0043] Hydrochloric acid, CAS No. 7647-01-0, molecular formula HCl, is prepared into a 1 mol / L aqueous solution using analytical grade hydrochloric acid and is used for pH adjustment of culture media.

[0044] Deionized water was used for both the preparation of the culture medium and the preparation of the reagent solutions.

[0045] Preparation Example 1: This preparation example provides a method for preparing a disease-inducing solid culture medium, including the following steps: Add 75% (v / v) of deionized water based on the final culture medium volume, add 1×MS basal medium and 2.5% (w / v) sucrose, and stir at 100 rpm to fully dissolve the basic nutrients to obtain the basal culture solution.

[0046] The obtained basic culture medium was heated to 45℃ and kept constant. PEG-4000 with a mass-volume concentration of 2.0% (w / v) and salicylic acid with a mass-volume concentration of 0.0015% (w / v) were added. The mixture was stirred continuously at 150 rpm for 25 min to fully disperse PEG-4000 and evenly distribute salicylic acid in the culture medium.

[0047] Maintain the culture medium temperature at 45℃, and slowly add potassium silicate aqueous solution to it. The added potassium silicate has a mass-volume concentration of 0.010% (w / v) based on SiO2, and the addition time is controlled to be 5 min. After the addition is complete, adjust the pH of the culture medium with 1 mol / L hydrochloric acid aqueous solution, and adjust the pH of the culture medium to 5.6 within 10 min.

[0048] Maintain the culture medium temperature at 45℃, add ammonium sulfate to make the ammonium sulfate concentration in the culture medium 0.066% (w / v), adjust the stirring speed to 100 rpm, and continue stirring for 12 min to fully disperse the ammonium sulfate.

[0049] Add deionized water to the obtained culture medium to make up the volume, and add agar powder with a mass-volume concentration of 0.6% (w / v). Stir at 100 rpm to make the agar powder evenly dispersed.

[0050] The obtained culture medium was dispensed into tissue culture containers and autoclaved at 121℃ and 0.1MPa for 20 minutes.

[0051] After sterilization, the culture medium was cooled to 68°C and isothermally incubated at 68°C for 45 minutes. After incubation, the culture medium was allowed to cool naturally at room temperature to allow the agar to gel, thus obtaining a disease resistance-inducing solid culture medium.

[0052] Preparation Example 2: This preparation example provides a method for preparing a disease-inducing solid culture medium, including the following steps: Based on the final culture medium volume, add 77.5% (v / v) of deionized water, 1×MS basal medium, and 3.0% (w / v) sucrose. Stir at 125 rpm to fully dissolve the basic nutrients and obtain the basal culture solution.

[0053] The obtained basic culture medium was heated to 50℃ and kept constant. PEG-4000 with a mass-volume concentration of 3.0% (w / v) and salicylic acid with a mass-volume concentration of 0.0020% (w / v) were added. The mixture was stirred continuously at 175 rpm for 30 min to fully disperse PEG-4000 and evenly distribute salicylic acid in the culture medium.

[0054] Maintain the culture medium temperature at 50℃, and slowly add potassium silicate aqueous solution to it. The added potassium silicate has a mass-volume concentration of 0.0125% (w / v) based on SiO2, and the addition time is controlled at 7.5 min. After the addition is complete, adjust the pH of the culture medium with 1 mol / L hydrochloric acid aqueous solution, and adjust the pH of the culture medium to 5.7 within 12.5 min.

[0055] Maintain the culture medium temperature at 50℃, add ammonium sulfate to make the ammonium sulfate concentration in the culture medium 0.198% (w / v), adjust the stirring speed to 125 rpm, and continue stirring for 15 min to fully disperse the ammonium sulfate.

[0056] Add deionized water to the obtained culture medium to make up the volume, and add agar powder with a mass-volume concentration of 0.7% (w / v). Stir at 125 rpm to make the agar powder evenly dispersed.

[0057] The obtained culture medium was dispensed into tissue culture containers and autoclaved at 121℃ and 0.1MPa for 22.5 min.

[0058] After sterilization, the culture medium was cooled to 70°C and isothermally incubated at 70°C for 52.5 min. After incubation, the culture medium was allowed to cool naturally at room temperature to allow the agar to gel, thus obtaining a disease resistance-inducing solid culture medium.

[0059] Preparation Example 3: This preparation example provides a method for preparing a disease-inducing solid culture medium, including the following steps: Add 80% (v / v) of deionized water based on the final culture medium volume, add 1×MS basal medium and 3.5% (w / v) sucrose, and stir at 150 rpm to fully dissolve the basic nutrients to obtain the basal culture solution.

[0060] The obtained basic culture medium was heated to 55℃ and kept constant. PEG-4000 with a mass-volume concentration of 4.0% (w / v) and salicylic acid with a mass-volume concentration of 0.0025% (w / v) were added. The mixture was stirred continuously at 200 rpm for 35 min to fully disperse PEG-4000 and evenly distribute salicylic acid in the culture medium.

[0061] Maintain the culture medium temperature at 55℃, and slowly add potassium silicate aqueous solution to it. The added potassium silicate has a mass-volume concentration of 0.015% (w / v) based on SiO2, and the addition time is controlled to be 10 min. After the addition is complete, adjust the pH of the culture medium with 1 mol / L hydrochloric acid aqueous solution, and adjust the pH of the culture medium to 5.8 within 15 min.

[0062] Maintain the culture medium temperature at 55℃, add ammonium sulfate to make the ammonium sulfate concentration in the culture medium 0.396% (w / v), adjust the stirring speed to 150 rpm, and continue stirring for 18 min to fully disperse the ammonium sulfate.

[0063] Add deionized water to the obtained culture medium to make up the volume, and add agar powder with a mass-volume concentration of 0.8% (w / v). Stir at 150 rpm to make the agar powder evenly dispersed.

[0064] The obtained culture medium was dispensed into tissue culture containers and autoclaved at 121℃ and 0.1MPa for 25 minutes.

[0065] After sterilization, the culture medium was cooled to 72°C and isothermally incubated at 72°C for 60 minutes. After incubation, the culture medium was allowed to cool naturally at room temperature to allow the agar to gel, thus obtaining a disease resistance-inducing solid culture medium. Example

[0066] This embodiment provides a method for tissue culture breeding of chili peppers with strong disease resistance, including the following steps: Pepper plants with consistent growth and no obvious diseases were selected. Cotyledons were used as explants. After surface disinfection, they were inoculated into primary culture medium under aseptic conditions to obtain sterile explants. The length of the explants was controlled to be 8 mm, and the number of explants was 30.

[0067] The obtained sterile explants were transferred to the disease resistance inducible solid culture medium obtained in Preparation Example 1 for induction culture. The culture temperature was controlled at 23℃, the daily light time was 14h, the dark time was 10h, the light intensity was controlled at 2000lx, and the culture time was 14d, so that the explants could complete the induction culture in a culture environment containing salicylic acid, PEG-4000 and silicon source.

[0068] After the induction culture was completed, the regenerated buds were transferred to the recovery culture medium for recovery culture. The recovery culture temperature was controlled at 23℃, the daily light time was 14h and the dark time was 10h, and the culture time was 10d to allow the regenerated buds to return to normal growth.

[0069] The regenerated shoots after recovery culture were transferred to rooting medium for rooting culture. The rooting culture temperature was controlled at 23℃, with a daily light time of 14h and a dark time of 10h, and the culture time was 15d to allow the regenerated shoots to form a complete root system and obtain pepper tissue culture seedlings.

[0070] The obtained chili tissue culture seedlings were subjected to hardening treatment at a temperature of 23℃ for 5 days. They were then transplanted into a cultivation substrate for further cultivation to obtain regenerated chili plants. Example

[0071] This embodiment provides a method for tissue culture breeding of chili peppers with strong disease resistance, including the following steps: Pepper plants with consistent growth and no obvious diseases were selected. Cotyledons were used as explants. After surface disinfection, they were inoculated into primary culture medium under aseptic conditions to obtain sterile explants. The length of the explants was controlled to be 10 mm, and the number of explants was 30.

[0072] The obtained sterile explants were transferred to the disease resistance inducible solid culture medium obtained in Preparation Example 2 for induction culture. The culture temperature was controlled at 25°C, the daily light time was 15h, the dark time was 9h, the light intensity was controlled at 2500lx, and the culture time was 21d, so that the explants could complete the induction culture in a culture environment containing salicylic acid, PEG-4000 and silicon source.

[0073] After the induction culture was completed, the regenerated buds were transferred to the recovery culture medium for recovery culture. The recovery culture temperature was controlled at 25℃, the daily light time was 15h and the dark time was 9h, and the culture time was 15d to allow the regenerated buds to return to normal growth.

[0074] The regenerated shoots after recovery culture were transferred to rooting medium for rooting culture. The rooting culture temperature was controlled at 25℃, with a daily light time of 15h and a dark time of 9h, and the culture time was 20 days, so that the regenerated shoots could form a complete root system and obtain pepper tissue culture seedlings.

[0075] The obtained chili tissue culture seedlings were hardened off at a temperature of 25℃ for 7 days, and then transplanted into a cultivation substrate for further cultivation to obtain regenerated chili plants. Example

[0076] This embodiment provides a method for tissue culture breeding of chili peppers with strong disease resistance, including the following steps: Pepper plants with consistent growth and no obvious diseases were selected. Cotyledons were used as explants. After surface disinfection, they were inoculated into primary culture medium under aseptic conditions to obtain sterile explants. The length of the explants was controlled to be 12 mm, and the number of explants was 30.

[0077] The obtained sterile explants were transferred to the disease resistance inducible solid culture medium obtained in Preparation Example 3 for induction culture. The culture temperature was controlled at 27°C, the daily light time was 16h, the dark time was 8h, the light intensity was controlled at 3000lx, and the culture time was 28d, so that the explants could complete the induction culture in a culture environment containing salicylic acid, PEG-4000 and silicon source.

[0078] After the induction culture was completed, the regenerated buds were transferred to the recovery culture medium for recovery culture. The recovery culture temperature was controlled at 27℃, the daily light time was 16h and the dark time was 8h, and the culture time was 20d to allow the regenerated buds to return to normal growth.

[0079] The regenerated shoots after recovery culture were transferred to rooting medium for rooting culture. The rooting culture temperature was controlled at 27℃, with a daily light time of 16h and a dark time of 8h, and the culture time was 25 days, so that the regenerated shoots could form a complete root system and obtain pepper tissue culture seedlings.

[0080] The obtained chili tissue culture seedlings were subjected to hardening treatment at a temperature of 27℃ for 10 days. They were then transplanted into the cultivation substrate for further cultivation to obtain regenerated chili plants.

[0081] Comparative Example 1: Compared with Example 2, the difference is that PEG-4000, salicylic acid, potassium silicate and ammonium sulfate are not added to the culture medium, and 1×MS basal medium, sucrose and agar powder are retained. The other culture medium preparation conditions and pepper tissue culture steps are the same as in Example 2.

[0082] Comparative Example 2: Compared with Example 2, the difference is that only salicylic acid was added to the culture medium, and PEG-4000, potassium silicate and ammonium sulfate were not added, while the other parameters and steps were the same.

[0083] Comparative Example 3: The difference from Example 2 is that PEG-4000 is not added to the culture medium, while the other components, amounts added, and preparation steps are the same as in Example 2.

[0084] Comparative Example 4: Compared with Example 2, the difference is that potassium silicate is not added to the culture medium, while the other components, addition amounts and preparation steps are the same as in Example 2.

[0085] Comparative Example 5: Compared with Example 2, the difference is that after sterilization, the 70°C isothermal heat treatment is not performed. Instead, the temperature is directly lowered to room temperature for gelation. All other parameters and steps are the same.

[0086] Comparative Example 6: Compared with Example 2, the difference is that the order of adding the components of the culture medium is changed. PEG-4000, salicylic acid, potassium silicate and ammonium sulfate are added to the basic culture medium at the same time, and the pH is adjusted to 5.7 after stirring evenly. All other parameters and steps are the same.

[0087] Test Example 1: Experimental description: This test was used to verify that the disease-resistant pepper tissue culture method described in Examples 1-3 can be implemented normally, and to evaluate the growth and regeneration of pepper explants under different implementation methods.

[0088] Pepper materials of consistent origin and similar growth stages were selected as experimental subjects and cultured according to the methods described in Examples 1, 2, and 3, respectively. Each example was performed in triplicate, with 30 explants inoculated in each replicate. Cultures were conducted under the conditions corresponding to Examples 1, 2, and 3, and observations and statistical analyses were performed. The time when sterile explants were transferred to disease-inducing solid culture medium to begin induction culture was defined as the culture start time.

[0089] Experimental steps: The aseptically treated pepper explants were cultured according to the methods corresponding to Examples 1, 2 and 3, and the changes in the state of the explants during the culture process were recorded. At 7, 14 and 28 days of culture, observations and statistics were conducted on each treatment group. The number of surviving explants, the number of explants that turned brown, the number of callus tissues formed and the number of regenerated shoots formed were recorded. The explant survival rate, explant browning rate, callus tissue formation rate and shoot differentiation rate were calculated. After completing the corresponding rooting culture in Examples 1, 2 and 3, the number of pepper tissue culture seedlings that formed complete root systems was counted, and the rooting rate was calculated. After completing the corresponding hardening treatment in Examples 1, 2 and 3 and transplanting them into the cultivation substrate for continued cultivation, the number of normally regenerated plants was counted and the normal regeneration rate was calculated.

[0090] Experimental data: The results of the explant growth and regeneration performance tests during the pepper tissue culture process in Examples 1-3 are shown in Table 1.

[0091] Table 1: Results of growth and regeneration performance tests of peppers during tissue culture.

[0092] Experimental conclusion: As shown in Table 1, the methods described in Examples 1-3 can all achieve pepper explant culture, callus formation, bud differentiation, rooting culture, and regenerated plant acquisition, indicating that the methods described in Examples 1-3 can all complete the pepper tissue culture regeneration process.

[0093] In the observations after 7, 14, and 28 days of culture, the explant survival rate of Example 2 was higher than that of Examples 1 and 3, the explant browning rate was lower than that of Examples 1 and 3, and the callus formation rate and bud differentiation rate were higher than those of Examples 1 and 3. At 28 days of culture, the explant survival rate of Example 2 was 92.8%, the explant browning rate was 10.8%, the callus formation rate was 84.6%, and the bud differentiation rate was 63.7%.

[0094] See attached document Figure 1 The bud differentiation rates of Examples 1, 2, and 3 all increased with prolonged culture time. After 7 days of culture, the bud differentiation rates of Examples 1, 2, and 3 were 26.8%, 32.5%, and 30.9%, respectively; after 14 days, they were 41.6%, 56.8%, and 53.2%; and after 28 days, they were 48.9%, 63.7%, and 59.4%. At each detection time point of 7, 14, and 28 days of culture, the bud differentiation rate of Example 2 was higher than that of Examples 1 and 3, and the bud differentiation rate of Example 3 was higher than that of Example 1. With prolonged culture time, the increase in bud differentiation rate of each example gradually decreased, with Example 2 maintaining a relatively high bud differentiation rate from 14 to 28 days of culture.

[0095] After completing the corresponding rooting culture in Examples 1, 2, and 3, the rooting rates were 82.4%, 88.9%, and 85.7%, respectively. After completing the corresponding hardening-off treatment and transplanting, the rates of obtaining normally regenerated plants were 39.5%, 52.4%, and 47.6%, respectively. The rooting rate and the rate of obtaining normally regenerated plants in Example 2 were both higher than those in Examples 1 and 3. Combining the test results of the above bud differentiation rate, rooting rate, and rate of obtaining normally regenerated plants, it can be seen that the culture conditions described in Examples 1-3 can all support the regeneration process of pepper tissue culture, among which the regeneration performance under the corresponding culture conditions in Example 2 was better.

[0096] Test Example 2: Experimental description: This test was used to compare the culture effects of Example 2 and Comparative Examples 1-6 in the process of pepper tissue culture, and to investigate the effects of different culture medium compositions and treatment conditions on pepper explant survival, browning, regenerated shoot formation, shoot elongation, rooting and the acquisition of normal regenerated plants.

[0097] Pepper explants of consistent origin and similar growth stages were selected as experimental materials. Example 2 involved cultivation according to the corresponding methods. Comparative Example 1 used a cultivation method without PEG-4000, salicylic acid, potassium silicate, and ammonium sulfate; Comparative Example 2 used a cultivation method with only salicylic acid added; Comparative Example 3 omitted PEG-4000; Comparative Example 4 omitted potassium silicate; Comparative Example 5 omitted isothermal incubation; and Comparative Example 6 changed the order of addition of the components of the culture medium. Each treatment group was set up in 3 independent replicates, with 30 explants inoculated in each replicate. Except for the culture medium composition or treatment conditions corresponding to each comparative example, all other culture conditions were kept consistent.

[0098] Experimental steps: Pepper explants of similar size and growth status were selected, and after treatment, they were inoculated into the culture media corresponding to Example 2 and Comparative Examples 1-6, respectively. Each treatment group was cultured under the same culture conditions, and explant survival, browning, regeneration of shoots and root growth were observed during the culture period. The growth status of explants in each treatment group was observed after 7 and 14 days of culture. After 28 days of culture, the survival rate, browning rate, average number of regenerated buds and average length of regenerated buds of each treatment group were statistically analyzed. After each treatment group completed the rooting culture, the number of pepper tissue culture seedlings that formed complete root systems was counted, and the rooting rate was calculated. After each treatment group completed the hardening-off treatment and transplanted the seedlings into the cultivation substrate for continued cultivation, the number of normally regenerated plants was counted, and the normal regeneration rate was calculated. Take uninoculated culture media from each treatment group, prepare culture media blocks of the same size under the same conditions, add an equal volume of deionized water as receiving solution, and place at 25°C for 24 hours. Collect the receiving liquid from each treatment group and perform ultraviolet-visible absorption spectroscopy in the range of 260–360 nm. Record the absorbance changes in the relevant absorption bands of salicylic acid and compare the differences in the curves of each treatment group.

[0099] Experimental data: The effects of chili tissue culture on Example 2 and Comparative Examples 1-6 are shown in Table 2.

[0100] Table 2: Comparison Test Results of Pepper Tissue Culture Effects

[0101] Note: Explant survival rate, explant browning rate, average number of regenerated shoots, and average length of regenerated shoots are statistical results after 28 days of culture; rooting rate is statistical result after the completion of rooting culture; normal regenerated plant acquisition rate is statistical result after the completion of hardening treatment and transplanting; data in the table are expressed as mean ± standard deviation.

[0102] Experimental conclusion: As shown in Table 2, the survival rate of explants after 28 days of culture in Example 2 was 92.8%, the browning rate of explants was 10.8%, the average number of regenerated buds was 4.7, and the average length of regenerated buds was 3.86 cm. The rooting rate after completing the rooting culture was 88.9%, and the rate of obtaining normal regenerated plants after completing the hardening treatment and transplanting was 52.4%.

[0103] Comparative Example 1, using a culture method without PEG-4000, salicylic acid, potassium silicate, and ammonium sulfate, showed lower regeneration indicators than Example 2. In Comparative Example 2, with salicylic acid added alone, the explant browning rate after 28 days of culture was 35.8%, and the rate of normal regenerated plants after hardening and transplanting was 22.9%. In Comparative Example 3, without PEG-4000, the explant browning rate after 28 days of culture was 29.7%, and the rate of normal regenerated plants after hardening and transplanting was 28.6%. These results indicate that neither adding salicylic acid alone nor removing PEG-4000 yielded the same culture effect as Example 2. The coexistence of PEG-4000 and salicylic acid helps reduce explant browning during culture and maintains the subsequent regeneration process.

[0104] See attached document Figure 2 Each treatment group exhibited varying degrees of absorption response around 298 nm. Comparative Example 2 showed the highest absorption peak in this band, followed by Comparative Example 3, with the absorption peaks of Comparative Examples 5, 6, and 4 decreasing sequentially. Example 2 also showed a corresponding absorption peak, but its peak value was lower than the aforementioned treatment groups, and the main absorption peak was relatively flat. Comparative Example 1, without the addition of salicylic acid, showed the lowest absorption response in this band. Considering the culture conditions of each treatment group, it was found that adding salicylic acid alone, removing PEG-4000, removing isothermal insulation, or changing the order of component addition all resulted in higher salicylic acid-related absorption responses in the receiving medium after 24 hours compared to Example 2. This indicates that the composition of the culture medium and the treatment process affect the migration of relevant components into the receiving medium during the initial stage of culture. Example 2 maintained the salicylic acid-related absorption response while exhibiting a lower absorption response in the initial stage of culture compared to the aforementioned treatment groups, which corresponds to the lower explant browning rate and higher explant survival rate shown in Table 2.

[0105] In Comparative Example 4, after removing potassium silicate, the rate of normal regenerated plants obtained after hardening and transplanting was 40.8%; in Comparative Example 5, after removing isothermal insulation treatment, the rate of normal regenerated plants obtained after hardening and transplanting was 36.2%; in Comparative Example 6, after changing the order of adding the components of the culture medium, the rate of normal regenerated plants obtained after hardening and transplanting was 33.5%, all of which were lower than in Example 2.

[0106] Combine Table 2 and Appendix Figure 2It is evident that the addition of PEG-4000, the presence of potassium silicate, the isothermal incubation treatment after sterilization, and the order of addition of each component of the culture medium all affect the culture results. Example 2, while reducing explant browning, exhibited higher average number of regenerated shoots, average length of regenerated shoots, rooting rate, and rate of obtaining normal regenerated plants, indicating that the components of the culture medium and the treatment conditions work together to facilitate the process of pepper explants completing the culture to form normal regenerated plants.

[0107] Test Example 3: Experimental description: This test was used to compare the changes in browning and oxidative damage-related indicators of pepper explants under the corresponding culture conditions of Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3, and to analyze the effects of different culture conditions on the culture status of pepper explants.

[0108] Pepper explants of consistent origin and similar growth stages were selected as experimental materials. Example 2 used the disease resistance-inducing solid culture medium corresponding to Example 2. Comparative Example 1 used a culture medium without PEG-4000, salicylic acid, potassium silicate, and ammonium sulfate. Comparative Example 2 used a culture medium with only salicylic acid. Comparative Example 3 used a culture medium based on the disease resistance-inducing solid culture medium corresponding to Example 2 without PEG-4000. Each treatment group was set up in three independent replicates, with the same number of explants used in each replicate, and all other culture conditions remained consistent.

[0109] In this test, pepper explants from each treatment group were cultured continuously in their respective culture media for 28 days without subsequent recovery culture, rooting culture, or hardening-off treatment. Observations and samples were taken at 3, 7, 14, and 28 days of culture to detect explant browning rate, H2O2 content, MDA content, PPO activity, POD activity, and SOD activity. H2O2 and MDA were used to evaluate the degree of oxidative damage to explants during culture, PPO was used to evaluate tissue browning-related changes, and POD and SOD were used to evaluate the antioxidant enzyme response of explants.

[0110] Experimental steps: Pepper explants with similar growth states were inoculated into the culture media corresponding to Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3, and cultured continuously for 28 days under the same environmental conditions. The explant status of each treatment group was observed at 3, 7, 14 and 28 days of culture. The number of explants that turned brown was counted and the browning rate of explants was calculated. Explants from each treatment group were selected at each detection time point, and equal amounts of fresh tissue were taken for homogenization and extraction. The H2O2 content and MDA content were determined respectively. Enzyme extracts were prepared from explant tissues of the same batch, and the activities of PPO, POD and SOD were measured respectively. The same sampling amount and detection conditions were maintained for each treatment group. Explants from each treatment group were harvested after 7 days of culture for MDA colorimetric reaction. Ultraviolet-visible absorption spectroscopy was scanned in the range of 450–650 nm, and the absorption response near 532 nm was compared. Data from each detection time point were compiled, and the culture damage of explants under different culture conditions was compared by combining the UV-Vis absorption curves at 7 days of culture.

[0111] Experimental data: Table 3 shows the browning and oxidative damage indices of pepper explants during 28 days of continuous culture under the culture conditions corresponding to Example 2 and Comparative Examples 1-3.

[0112] Table 3: Test Results of Oxidation Damage Related Indicators

[0113] Note: Data in the table are expressed as average values, and each treatment group was set up with 3 independent replicates.

[0114] Experimental conclusion: As shown in Table 3, the H2O2 content of the pepper explants under the culture conditions corresponding to Example 2 was 2.84 μmol·g at 3, 7, 14, and 28 days of culture. -1 FW, 3.41 μmol·g -1 FW, 3.12 μmol·g -1 FW and 2.67 μmol·g - 1 The FW and MDA contents were 8.6 nmol·g. -1 FW, 10.9 nmol·g -1 FW, 10.1 nmol·g -1 FW and 9.3 nmol·g -1 FW. Both of the above indicators increased after 7 days of culture, and then gradually decreased. In Example 2, under the corresponding culture conditions, POD and SOD activities remained at a certain level during culture, and the explant browning rate was 10.8% after 28 days of culture. In contrast, in Comparative Example 2, the H2O2 content reached 7.46 μmol·g after 7 days of culture. -1 FW, MDA content reaches 24.7 nmol·g -1 FW, PPO activity is 52.8 U·g -1In the case of FW, the browning rate of explants increased to 21.8%; after 28 days of culture, the browning rate of explants in Comparative Example 2 further increased to 35.8%. The above results indicate that when salicylic acid is added alone, the H2O2 content, MDA content, and PPO activity of explants in the early stage of culture are all higher than those in the corresponding culture conditions of Example 2, accompanied by a higher browning rate of explants.

[0115] In Comparative Example 3, without the addition of PEG-4000, the H2O2 content was 6.19 μmol·g after 7 days of cultivation. -1 FW, MDA content is 20.6 nmol·g -1 FW, PPO activity is 45.2 U·g -1 The browning rate (FW) of explants in Comparative Example 3 was higher than that in Example 2 but lower than that in Comparative Example 2. After 28 days of culture, the browning rate of explants in Comparative Example 3 was 29.7%, higher than that in Example 2. Combined with the results of POD and SOD activity tests, it can be seen that both Comparative Example 2 and Comparative Example 3 showed increased antioxidant enzyme activity in the early stages of culture, while H2O2 content, MDA content, and PPO activity were higher than those in Example 2. Under the culture conditions corresponding to Example 2, H2O2 content, MDA content, PPO activity, and explant browning rate remained at relatively low levels, indicating that the presence of PEG-4000 in conjunction with salicylic acid and other culture medium components can reduce oxidative damage and tissue browning in explants during the early stages of culture.

[0116] See attached document Figure 3 In all four treatment groups, the MDA colorimetric reaction solution formed a major absorption peak near 532 nm. The peak values, from highest to lowest, were Comparative Example 2, Comparative Example 3, Comparative Example 1, and Example 2, consistent with the order of MDA content changes after 7 days of culture in Table 3. The absorption responses near 532 nm in Comparative Example 2 and Comparative Example 3 were higher than those in Example 2, indicating that the addition of salicylic acid alone and the removal of PEG-4000 resulted in higher levels of membrane lipid peroxidation in the explants after 7 days of culture. Combined with the changes in H2O2 content, PPO activity, and explant browning rate, it can be seen that Example 2 can control oxidative damage in the early stages of culture while retaining the effect of salicylic acid, maintaining a relatively stable antioxidant enzyme response and a low browning level in the explants, thus providing a more suitable culture state for subsequent bud differentiation, rooting, and regenerated plant formation.

[0117] Test Example 4: Experimental description: This test was used to compare the changes in salicylic acid content and soluble silicon content (calculated as SiO2) during the cultivation of Examples 2, 3, 4, 5 and 6, and to analyze the effects of PEG-4000, potassium silicate, isothermal incubation treatment and the order of addition of each component of the culture medium on the changes in salicylic acid content and soluble silicon content (calculated as SiO2) during the cultivation process.

[0118] Pepper explants of consistent origin and similar growth stages were selected as experimental materials and cultured under the conditions corresponding to Examples 2, 3, 4, 5, and 6. Each treatment group was independently replicated three times, and culture medium samples were collected at 3, 7, and 14 days to determine the salicylic acid content and the soluble silicon content (calculated as SiO2). Except for differences in culture medium composition or corresponding treatment conditions, all other culture and detection conditions remained consistent across treatment groups.

[0119] Experimental steps: Pepper explants with similar growth states were inoculated into the culture media corresponding to Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6, and cultured under the same environmental conditions. Equal amounts of culture medium samples were taken from each treatment group at 3, 7 and 14 days of culture, and the test solutions were obtained after extraction and filtration under the same conditions. The content of salicylic acid in the test solution was detected by high performance liquid chromatography, and the same extraction conditions, injection volume and chromatographic detection conditions were maintained for each treatment group. Another culture medium sample taken at the same time point was used to extract water-soluble components, and the soluble silicon content in the test solution, calculated as SiO2, was determined by the silicomolybdenum blue spectrophotometric method. Data from each detection time point were compiled to compare the changes in salicylic acid content and soluble silicon content (calculated as SiO2) in different treatment groups with culture time.

[0120] Experimental data: The changes in salicylic acid content and soluble silicon content (calculated as SiO2) during the cultivation process of different treatment groups are shown in Table 4.

[0121] Table 4: Test results of salicylic acid and soluble silicon content

[0122] Experimental conclusion: Table 4 shows that the salicylic acid content in all treatment groups decreased with increasing culture time, but the rate of decrease varied among the different treatment groups. In Example 2, the salicylic acid content was 18.7 mg / L at 3, 7, and 14 days of culture. -1 15.9 mg·L -1 and 12.6 mg·L -1 The salicylic acid content of Comparative Examples 3, 5, and 6 after 3 days of culture was 20.8 mg·L⁻¹. -1 21.3 mg·L -1 and 20.5 mg·L -1The levels were all higher than in Example 2; after 14 days of culture, the salicylic acid content in Comparative Examples 3, 5, and 6 decreased to 7.6 mg·L⁻¹. -1 8.9 mg·L -1 and 8.2 mg·L -1 The levels were all lower than in Example 2. In Comparative Example 4, the salicylic acid content at 3, 7, and 14 days of culture was 18.1 mg·L⁻¹. -1 15.2 mg·L -1 and 11.8 mg·L -1 .

[0123] Regarding the soluble silicon content, calculated as SiO2, in Example 2, the content was 118.4 mg·L⁻¹ at 3, 7, and 14 days of cultivation. -1 103.7 mg·L -1 and 86.9 mg·L -1 Comparative Examples 3, 5, and 6 had a concentration of 62.8 mg / L after 14 days of culture. -1 55.4 mg·L -1 and 49.7 mg·L -1 The values ​​were all lower than those in Example 2. In Comparative Example 4, without the addition of potassium silicate, the soluble silicon content (based on SiO2) was 13.7 mg·L⁻¹ at 3, 7, and 14 days of cultivation. -1 9.6 mg·L -1 and 6.8 mg·L -1 The values ​​were all lower than in Example 2. These results indicate that potassium silicate is the main source of soluble silicon in the culture system; without the addition of PEG-4000, without isothermal incubation, or by changing the order of addition of the components of the culture medium, the changes in salicylic acid content and soluble silicon content (calculated as SiO2) with culture time differed from those in Example 2.

[0124] See attached document Figure 4 The trends of salicylic acid content and soluble silicon content (calculated as SiO2) in each treatment group with culture time were consistent with the detection results in Table 4. Sub-figure A shows that the salicylic acid content of Example 2 was lower than that of Comparative Examples 3, 5, and 6 after 3 days of culture; however, the salicylic acid content of Example 2 was higher than that of Comparative Examples 3, 5, and 6 after 7 and 14 days of culture. In Comparative Example 3, without the addition of PEG-4000; in Comparative Example 5, without isothermal incubation; and in Comparative Example 6, after changing the order of addition of the culture medium components, the salicylic acid content was higher than that of Example 2 after 3 days of culture, but lower than that of Example 2 after 7 and 14 days of culture.

[0125] Subfigure B shows that in Example 2, the soluble silicon content (based on SiO2) was higher than that in Comparative Examples 3, 5, and 6 at 3, 7, and 14 days of culture. In Comparative Example 4, without the addition of potassium silicate, the soluble silicon content (based on SiO2) was lower than that of the other treatment groups at all three detection time points. Combining the changes in Subfigures A and B, it can be seen that without the addition of PEG-4000, without isothermal incubation, or by changing the order of addition of the components of the culture medium, the salicylic acid content and the soluble silicon content (based on SiO2) changed with culture time.

[0126] Summary Table 4 and Appendix Figure 4 As a result, the salicylic acid content of Example 2 was lower than that of Comparative Examples 3, 5 and 6 after 3 days of cultivation, while the salicylic acid content was higher than that of Comparative Examples 3, 5 and 6 after 7 and 14 days of cultivation; the soluble silicon content of Example 2, calculated as SiO2, was higher than that of Comparative Examples 3, 5 and 6 after 3, 7 and 14 days of cultivation.

[0127] Combining the results of Test Examples 2 and 3, the changes in salicylic acid content and soluble silicon content (calculated as SiO2) with culture time in Example 2 corresponded to a lower explant browning rate, lower H2O2 content and MDA content, and higher regeneration and rooting indices. These results indicate that PEG-4000, potassium silicate, isothermal incubation treatment, and the order of addition of the culture medium components all affect the changes in salicylic acid content and soluble silicon content (calculated as SiO2) during culture. Under the culture conditions corresponding to Example 2, the content changes of salicylic acid and soluble silicon (calculated as SiO2) remained different from other treatment groups during culture days 3–14.

[0128] Test Example 5: Experimental description: This test case is used to compare the disease occurrence of pepper regenerated plants obtained from Example 2, Comparative Example 1 and Comparative Example 2 after inoculation with pepper anthracnose fungus, and to compare the leaf condition of pepper regenerated plants in different treatment groups after pathogen inoculation by using the visible and near-infrared reflectance spectra of leaves.

[0129] Experimental steps: Pepper regenerated plants of the same age and similar growth status obtained from Example 2, Comparative Example 1 and Comparative Example 2 were selected. Each treatment group was set up with 3 independent replicates, with 20 plants in each replicate. To prepare a suspension of anthracnose spores of pepper, all treatment groups used the same concentration of anthracnose spore suspension and inoculated the leaves of the regenerated pepper plants of each treatment group using the same inoculation method. After inoculation, the culture environment was kept consistent. The occurrence of diseases in the regenerated pepper plants of each treatment group was observed at 3, 7 and 12 days after inoculation. The degree of disease occurrence in each treatment group was recorded and the disease index was calculated according to the same disease investigation method. Seven days after inoculation, complete leaves at the same position in each treatment group were selected, and the leaf reflectance spectrum in the range of 400-1000 nm was measured under the same detection conditions. The same light source, probe distance and integration time were used for each treatment group, and the average value of the repeated detection results was taken. Twelve days after inoculation, the number of diseased plants in each treatment group was counted and the incidence rate was calculated. The disease index, incidence rate, and leaf reflectance spectral characteristics of different treatment groups were compared.

[0130] Experimental data: Table 5 shows the disease occurrence and leaf reflectance spectral characteristics of regenerated pepper plants in different treatment groups after inoculation with pepper anthracnose.

[0131] Table 5: Anthracnose resistance and leaf reflectance spectrum test results of regenerated pepper plants

[0132] Experimental conclusion: As shown in Table 5, the disease indices of the regenerated pepper plants obtained in Example 2 were 2.8, 8.3, and 12.1 at 3, 7, and 12 days after inoculation with *Anthracnose*, respectively, and the incidence rate was 19.4% at 12 days after inoculation. The disease indices of the regenerated pepper plants obtained in Comparative Example 1 were 4.7, 18.6, and 36.8 at the same time points, and the incidence rate was 51.7% at 12 days after inoculation; the disease indices of the regenerated pepper plants obtained in Comparative Example 2 were 5.3, 24.5, and 47.6 at the same time points, and the incidence rate was 62.3% at 12 days after inoculation.

[0133] As the inoculation time increased, the disease index of all three treatment groups increased. The disease index of the regenerated pepper plants obtained in Example 2 was lower than that of Comparative Examples 1 and 2 at 3, 7, and 12 days after inoculation, and the incidence rate at 12 days after inoculation was also lower than that of Comparative Examples 1 and 2. These results indicate that the regenerated pepper plants obtained in Example 2, after inoculation with *Anthracnose*, exhibited a lower degree of disease incidence than the regenerated pepper plants obtained in Comparative Examples 1 and 2, demonstrating better resistance to anthracnose.

[0134] See attached document Figure 5The reflectance spectra of the leaves in the three treatment groups showed different variation characteristics in the range of 400–1000 nm. The reflectance of the leaves of the regenerated pepper plants obtained in Example 2 was 0.044 at 680 nm, which was lower than 0.061 in Comparative Example 1 and 0.089 in Comparative Example 2; the reflectance at 750 nm was 0.476, which was higher than 0.394 in Comparative Example 1 and 0.282 in Comparative Example 2; and the reflectance at 850 nm was 0.503, which was higher than 0.441 in Comparative Example 1 and 0.361 in Comparative Example 2.

[0135] Appendix Figure 5 The changes in reflectance spectrum shown are consistent with the reflectance detection results at 680nm, 750nm and 850nm in Table 5, indicating that there are differences in the visible light and near-infrared reflectance characteristics of the leaves of the regenerated pepper plants in the three treatment groups after inoculation with pepper anthracnose.

[0136] Based on the combined results of disease surveys and leaf reflectance spectroscopy, the regenerated pepper plants obtained in Example 2, after inoculation with pepper anthracnose, exhibited lower disease index and incidence rates. Simultaneously, the leaves showed lower reflectance at 680 nm and higher reflectance at 750 nm and 850 nm. The leaf reflectance spectroscopy results corresponded to the changes in disease index and incidence rate.

[0137] Based on the results of Test Examples 2-4, the pepper explants under the culture conditions corresponding to Example 2 exhibited lower browning rates and oxidative damage-related indicators. During the culture process, the salicylic acid content and soluble silicon content (calculated as SiO2) maintained their corresponding changes, and the plants showed good regeneration and rooting performance. The disease index and incidence rate of the pepper regenerated plants obtained in Example 2 after inoculation with pepper anthracnose were lower than those obtained in Comparative Examples 1 and 2, indicating that the culture method described in Example 2 can obtain pepper regenerated plants with good anthracnose resistance.

[0138] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for tissue culture breeding of disease-resistant chili peppers, characterized in that, Includes the following steps: Cotyledons of chili plants were selected as explants. The explants were surface disinfected and then inoculated into a primary culture medium under aseptic conditions to obtain sterile explants. The sterile explants were transferred to a disease-inducing solid culture medium for induction culture to obtain regenerated shoots; The regenerated buds were transferred to a recovery culture medium for recovery culture to obtain regenerated buds after recovery culture. The regenerated shoots after recovery culture were transferred to a rooting medium for rooting culture to obtain pepper tissue culture seedlings; The chili tissue culture seedlings were hardened off and then transplanted into a cultivation substrate for further cultivation to obtain regenerated chili plants. The disease resistance-inducing solid culture medium comprises 1×MS basal medium, sucrose with a mass-volume concentration of 2.5%–3.5%, PEG-4000 with a mass-volume concentration of 2.0%–4.0%, salicylic acid with a mass-volume concentration of 0.0015%–0.0025%, potassium silicate with a mass-volume concentration of 0.010%–0.015% (based on SiO2), ammonium sulfate with a mass-volume concentration of 0.066%–0.396%, and agar powder with a mass-volume concentration of 0.6%–0.8%. The pH of the disease resistance-inducing solid culture medium is 5.6–5.

8.

2. The method for tissue culture of disease-resistant chili peppers according to claim 1, characterized in that, The disease resistance-inducing solid culture medium was prepared according to the following method: Add deionized water according to the final culture medium volume, then add 1×MS basal medium and sucrose and stir to obtain the basal culture medium; The basic culture medium was heated and kept at a constant temperature. PEG-4000 and salicylic acid were added and stirred to obtain the culture medium. Maintain the temperature of the culture medium, and add an aqueous solution of potassium silicate as the form of potassium silicate addition to the culture medium, wherein the amount of potassium silicate used is calculated according to the mass of SiO2 contained in the aqueous solution of potassium silicate; After the potassium silicate aqueous solution is added, the pH of the culture medium is adjusted using hydrochloric acid aqueous solution; Maintain the temperature of the culture medium, add ammonium sulfate to the culture medium and stir; Add deionized water to the culture medium after adding ammonium sulfate to make up the volume, then add agar powder and stir. The culture medium after adding the agar powder is dispensed into tissue culture containers, and the culture medium in the tissue culture containers is sterilized by autoclaving. After high-pressure steam sterilization, the culture medium in the tissue culture container is cooled and isothermally incubated. After isothermally incubation, the culture medium in the tissue culture container is allowed to cool naturally at room temperature to obtain the disease-inducing solid culture medium.

3. The method for tissue culture breeding of disease-resistant chili peppers according to claim 2, characterized in that, When preparing the basic culture medium: Based on the final culture medium volume, the deionized water accounts for 75% to 80% of the final culture medium volume; The stirring speed after adding the 1×MS basal medium and the sucrose was 100-150 rpm.

4. The method for tissue culture breeding of disease-resistant chili peppers according to claim 2, characterized in that, When the PEG-4000 and the salicylic acid are added to the basic culture medium: The basic culture medium is heated to 45–55°C and kept at a constant temperature. After adding the PEG-4000 and the salicylic acid, stir continuously at 150-200 rpm for 25-35 min.

5. The method for tissue culture breeding of disease-resistant chili peppers according to claim 2, characterized in that, When the potassium silicate aqueous solution is added to the culture medium and the pH is adjusted: When the potassium silicate aqueous solution is added, the temperature of the culture medium is maintained at 45-55°C; The potassium silicate aqueous solution is added over a period of 5 to 10 minutes. After the potassium silicate aqueous solution is added, the pH of the culture medium is adjusted with 1 mol / L hydrochloric acid aqueous solution for 10-15 min.

6. The method for tissue culture of disease-resistant chili peppers according to claim 2, characterized in that, When ammonium sulfate is added to the culture medium: The temperature of the culture medium is maintained at 45–55°C; After adding the ammonium sulfate, adjust the stirring speed to 100-150 rpm and continue stirring for 12-18 minutes.

7. The method for tissue culture of disease-resistant chili peppers according to claim 2, characterized in that, When adding the agar powder, performing the autoclaving, and performing the isothermal insulation: Add deionized water to the culture medium after adding ammonium sulfate to make up the volume, then add the agar powder, and stir at 100-150 rpm after adding the agar powder; When the culture medium dispensed into the tissue culture container is subjected to high-pressure steam sterilization, the high-pressure steam sterilization temperature is 121°C, the pressure is 0.1 MPa, and the time is 20-25 min. After the high-pressure steam sterilization is completed, the culture medium in the tissue culture container is cooled to 68-72°C and isothermally incubated at 68-72°C for 45-60 minutes.

8. The method for tissue culture of disease-resistant chili peppers according to claim 1, characterized in that, When the cotyledons of the pepper plant material are selected as explants and the explants are inoculated into the primary culture medium: When the cotyledons of the pepper plant material are selected as the explant, the length of the explant is 8-12 mm; When the explants are inoculated into the primary culture medium under aseptic conditions, the number of explants inoculated is 30.

9. The method for tissue culture of disease-resistant chili peppers according to claim 1, characterized in that, When transferring the sterile explants to the disease-resistant inducing solid culture medium for the induction culture: The sterile explants were transferred to the disease resistance-inducing solid culture medium for induction culture at a temperature of 23–27°C, with a daily light duration of 14–16 h and a dark duration of 8–10 h, and the daily light and dark duration totaled 24 h. The light intensity was 2000–3000 lx, and the culture time was 14–28 days.

10. The method for tissue culture of disease-resistant chili peppers according to claim 1, characterized in that, When performing the recovery culture on the regenerated buds, the rooting culture on the regenerated buds after recovery culture, and the hardening-off treatment on the pepper tissue culture seedlings: When the regenerated buds are transferred to the recovery culture medium for recovery culture, the recovery culture temperature is 23-27°C, the daily light time is 14-16 hours, the daily dark time is 8-10 hours, and the daily light and dark time is 24 hours, and the culture time is 10-20 days. When the regenerated shoots after the recovery culture are transferred to the rooting medium for rooting culture, the rooting culture temperature is 23-27℃, the daily light time is 14-16h, the dark time is 8-10h, and the culture time is 15-25d. When the chili tissue culture seedlings are subjected to the hardening treatment, the hardening temperature is 23-27℃ and the hardening time is 5-10 days.

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